1. Interfaces in Chromatography
SHIKHA D. POPALI
HARSHPAL SINGH WAHI

2. LC-MS interface

3. LC-MS
► LC – Separation of the mixture of analytes
► Interface – Separation of the analyte from the solvent
► MA (mass analyzer) – separation of the analyte
molecular ion and fragments according to their mass
to charge ratio
Interface
Mass
Analyzer
DetectorLC
Extraction of
The analyte from
The solvent
Ion evaporation
or ionization.
Fragmentation

4. PROBLEMS IN COMBINING
HPLC AND MS
HPLC
 Liquid phase operation
 25 - 50°C
 No mass range
limitations
 Inorganic buffers
 1 ml/min eluent flow is
equivalent to 500 ml/min
of gas
MS
 Vacuum operation
 200 - 300°C
 Up to 4000 Da for
quadrupole MS
 Requires volatile
buffers
 Accepts 10 ml/min gas
flow

5. Coupling methods
► Direct liquid introduction (DLI) reduces the flow entering the MS
using some kind of splitting device.
► Particle beam (PB)
► Moving belt/wire rely on removal of the solvent prior to entering
the MS.
► Continuous flow fast atom bombardment (cf-FAB)
► A serious drawback of the first approach is the reduction in
sensitivity caused by the split factor.
► The entire flowrate, about 1 mL/min, used with a classic 4.6 mm
i.d. column is only tolerated by techniques such as thermospray
(TSP) and atmospheric pressure chemical ionization (APCI).
► Electrospray (ES) has a working range from nL/min to 0.2 mL/min,
which can be extended to mL/min flows.

6. Direct Liquid Introduction
Scheme of the DLI interface. 1 connection to LC column, 2 diaphragm 5
μm opening to MS, 3 needle valve, 4 cooling region, 5 to UV detector
or waste.

7. ► The first attempts to introduce a liquid into an MS using the
classic electron impact ionization (EI)/chemical ionization
(CI) source were based on the simple principle that by
minimizing the amount of liquid,
► the vacuum system would remove the solvent leaving the
analyte in the gas phase for ionization.
► By using larger pump systems and differential pumping,
maintenance of the vacuum was ensured.
► The flow from the LC column was reduced by using smaller i.d.
columns and/or splitting the liquid flow. In order to assist the
evaporation a heated de-solvation chamber could be
introduced

8. Particle beam
Schematic showing the principal components of a particle beam or MAGIC
interface

9. ► Ionization in most LC–MS interfaces depends in some way on the
composition of the LC solvent.
► A interface that provides the possibility of using EI/CI without the
mechanical transport was named MAGIC, an acronym for mono
disperse aerosol generation interface for chromatography. This
system is now known as particle beam.
► The LC eluent is forced through a small nebulizer using a He gas
flow to form a stream of uniform droplets. These droplets move
through a de-solvation chamber and evaporate to a solid particle.
► These particles are separated from the gas/solvent and
transported into the MS source using a differentially pumped
momentum separator.
► The PB interface allows flow-rates from 0.1–0.5 mL/min. Most
analytes that are amenable to PB LC–MS can be analysed using
GC–MS as well.

10. Moving Belt/Wire
Schematic showing the principal components of a moving-belt interface

11. ► The moving-belt interface separates the condensed liquid-phase
side of the LC from the high vacuum of the MS and uses a belt to
transport the analytes from one to the other.
► The mobile phase of the LC is deposited on a band and
evaporated. The analytes remain on the continuously cycling belt
and are transported from atmospheric pressure into the vacuum
of the ion source through two differentially pumped vacuum
locks.
► A heater in the ion source evaporates the sample from the belt
allowing MS analysis.
► Most moving-belt analyses deal with volatile analytes using CI/EI;
however, less volatile molecules such as nucleosides and
nucleotides are analysed using this system.

12. Continuous flow fast atom bombardment
(cf-FAB)
Basic elements of a cf-FAB probe

13. ► Continuous-flow or dynamic FAB is a modification of the FAB
technique that allows continuous on-line refreshing of the liquid
on the FAB target.
► This liquid, a high-boiling solvent such as glycerol, thioglycerol or
nitrobenzyl alcohol, is added to the LC effluent and transported
through a capillary to the probe tip.
► There, fast atoms (often Ar or Xe, keV energies) bombard the
sample and ions are sputtered out of the solution and into the gas
phase, and sampled in the MS.
► Together with the sample some of the matrix is ionized as well as
yielding a pronounced chemical background at low m/z; however,
this phenomenon is more pronounced in static FAB.
► The most successful applications of cf-FAB are found in areas in
which highly polar, thermolabile, often involatile compounds are to
be analysed (surfactants, DNA adducts, oligonucleotides,
pesticides, pharmaceuticals etc.) because highly polar and/or ionic
species are successfully analysed using FAB.

14. Thermospray
Thermospray interface. (a) configuration for ‘real-TSP-ionization’
(filament off) or external ionization (filament on) (b)
configuration with discharge electrode for external ionization and
repeller electrode

15. ► The TSP interface was developed by M. Vestal and co–workers. A
major advantage of TSP over other LC–MS interfaces is its ability to
handle the high flow-rates delivered by LC (up to 2 mL/min).
► As the name thermospray implies, heating the liquid flow leaving an LC
system creates a spray of superheated mist containing small liquid
droplets.
► Several techniques are developed to heat and vaporize the effluent;
however, the most successful method involves directing the liquid flow
through an electrically heated capillary, which can be directly introduced
into the MS ion source.
► The droplets are further vaporized as they collide against the walls of
the heated ion source.
► This ion source is equipped with a mechanical pump line opposite to
the spray in order to evacuate the excess solvent vapour
► The rapid heating and protective effects of the solvent allow the
analysis of non-volatile samples without pyrolysis.

16. ► The analyte ions are sampled into the MS through a sampling cone, if
necessary aided by an applied electric field (repeller or accelerating
electrode).
► Ionization of the analytes in TSP occurs by means of several processes
wherein two classes of ionization can be distinguished:
► one without an external ionization source, called ‘real thermospray’
► one with an external ionization.
► The real thermospray uses a volatile buffer, often ammonium acetate, as
part of the LC effluent. The buffer ions NH4 and CH3COO act as chemical
ionization reagent ions to form protonated or deprotonated ions,
respectively.
► The basicity or acidity of the analytes relative to the buffer determines
which ions will or will not be produced. This ionization takes place both in
the gas phase after evaporation of the respective reagents, or in the
droplets.
► Ions formed in the liquid phase are subsequently transferred to the gas
phase either by evaporation of the solvent or by ion evaporation in which
ions are liberated immediately from the droplet.

17. ► When no buffer is used, or if a higher percentage of organic
modifier is employed, an external ionization is applied.
► In the TSP source, an electron-impact filament can be used to
induce a plasma of ions from the mobile phase. Alternatively, a
discharge electrode can be used to generate this plasma. These
modes, filament-on and discharge-on, yield solvent-mediated CI
spectra.
► Each ionization mode, positive or negative, yields molecular ions
(protonated/deprotonated or adducts with other ions).
► Depending on the nature of the analyte and the experimental
settings of source temperature and repeller voltage, some
fragmentation will occur.
► Although being a high-temperature technique, it has been
successfully applied to thermolabile compounds such as DNA
adducts, which cannot be analysed with, for example, GC–MS.

18. ► The earliest LC–MS techniques (DLI, TSP, moving belt, PB),
although commercialized, were often difficult to use, had
limited sensitivity and were not robust; however, they were
very useful for specific applications.
► The overwhelming increase in LC–MS applications is mainly
the result of the sensitivity and ruggedness of atmospheric
pressure ionization (API) LC–MS techniques.

19. ► API is a general name for all ionization techniques in which the
ions are formed at atmospheric pressure.
► Though very popular today, ionization processes at atmospheric
pressure (flames, discharges etc.) have been studied using mass
spectrometers
► In modern LC–MS applications; two major techniques: ES and
APCI.
► Electrospray can be subdivided into techniques such as
pneumatic-assisted ES, ES, multiple sprayer ES etc., that differ
mainly in the formation of a spray from the LC flow.
► However, all ES variants rely on the same mechanism(s) to form
ions from the droplets at atmospheric pressure.
► The ions formed at atmospheric pressure are transported from
the source to the vacuum of the analyser through one or more
differentially pumped stages separated by skimmers. The ions
are focused and guided through the skimmer openings into the
MS by applying appropriate electric fields.

20. ► Where ES has its optimal performance at low flow-rates (nL/min
range) APCI operates happily using mL/min flow-rates.
► ES and APCI perform differently under different chromatographic
modes.
► The advantages of API were summarized by Voyksner in four
points:
1. “API approaches can handle volumes of liquid typically used in
LC”
2. “API is suitable for the analysis of nonvolatile, polar and thermally
unstable compounds typically analysed by LC”
3. “API-MS systems are sensitive, offering comparable or better
detection limits than achieved by GC–MS”
4. “API systems are very rugged and relatively easy to use.”

21. Electrospray
Basic elements of an electrospray and the layout of a commercial
(Micromass) ES-source and probe.

22. Droplet and ion production under ES conditions.

23. ► In MS research, ES is also described as a sample application
method for plasma desorption
► The ES source comprises two electrodes; that is, the ES capillary
and the atmospheric pressure aperture plate of the mass
spectrometer as the counter electrode.
► A high-voltage supply maintains a potential difference of about 3
kV between both electrodes. Under influence of the applied electric
field, ions of the same polarity migrate toward the liquid at the
capillary tip, where the liquid surface is drawn out of the capillary
forming a ‘Taylor cone’.
► When the build-up of an excess of ions of one polarity at the
surface of the liquid reaches the point that coulombic forces are
sufficient to overcome the surface tension of the liquid, droplets (1
μm) enriched in one ion polarity are emitted from the capillary.
► These droplets shrink by solvent evaporation and repeated
disintegration, leading to very small (3–10 nm) charged droplets
capable of producing gas-phase ions, yielding a very soft
ionization technique.

24. ► The ions are sampled through a set of skimmer electrodes and finally
analysed in the MS analyser.
► In order to reduce detrimental effects of deposits on electrodes and
skimmers, and to avoid neutrals entering the MS vacuum, a gas curtain
is applied effectively blowing neutrals and large particles away from
the MS entrance and/or sprayer, and electrodes are set up such that
the ions must round one or more angles guided by the electric fields.
► ES ionization can be used for small molecules and low-polarity
compounds providing there is some way to (de)protonate or form a
combination with a cation or anion.
► However, the most striking application is that of high molecular weight,
thermolabile, polar biomolecules such as peptides, proteins,
oligonucleotides etc.,
► Another highly uncommon characteristic of ES is its ‘softness’; that is,
very labile structures can be carried as ions into the gas phase without
disrupting their structures. ES can be used to study protein folding
status, non-covalent bonding, DNA duplexes etc.
► For the same reason ES spectra contain little or no structural
information because of the absence of fragmentation.

25. Atmospheric Pressure Ionization
Overview of a differentially pumped API source coupled to a mass spectrometer

26. Atmospheric Pressure Chemical Ionization
► In APCI the liquid flow from the LC is sprayed and rapidly
evaporated by a coaxial nitrogen stream and heating the nebulizer
to high temperature (350–500 °C).
► Although these temperatures may degrade the analytes, the high
flow-rates and coaxial N2-flow prevent breakdown of the
molecules.
► Ions already present in solution can be carried into the gas phase,
however, additional ionization is achieved using a corona
discharge (3–6 kV) in the spray.
► This discharge can ionize not only the analyte molecules, but also
the solvent molecules. These solvent ions can react with the
analytes in the gas phase in the same way samples are ionized in
a CI source by the reagent gas.
► In positive ion mode protonated molecules and adducts are
formed; in negative ion mode ions are formed by deprotonation,
combinations with anions or electron-capture.

27. ► Unlike in ES, the solvent-evaporation and ion-formation processes are
separated in APCI. This allows the use of solvents that are
unfavourable for ion formation.
► These low-polarity solvents are commonly used in normal-phase
chromatography with low polarity samples that can generally be
evaporated for APCI ionization.
► Another major difference between APCI and ES can be found in the LC
flow-rates that are used. APCI is a technique with optimal performance
at high flow-rates (1 mL/min and higher).
► Lower flow-rates can be used; however, when flow-rates are too low
the stability of the corona discharge may become problematic.
Whereas ES is ideally suited to miniaturization, reducing the flows and
LC dimensions using APCI
► APCI finds most of its applications in molecular weights below 1000 Da
for medium- to low-polarity molecules. The analytes will need some
degree of volatility and should not be too thermolabile. Typical
molecules are pesticides, drugs, steroids, PAHs etc.
► The applications can be found in a variety of places, from quality
control, environmental monitoring (water, air, soil), geological studies,
metabolite studies etc.

28. ATMOSPHERIC PRESSURE PHOTOSPRAY
IONISATION SOURCE
 Uses UV lamp for ionisation (10eV)
 Uses toluene as “dopant” (ionised by UV to form photo ions), co-vaporized. Other
dopant acetone, anisole and chlorobenzene , isoprene
 Evaporation in a heated probe, similar to APCI
 Ion-molecule reactions initiated by the photoions
 Proton transfer analytes ionised as [M+H]+
 Charge transfer analytes ionised as [M]+•

29. GC-MS interface

30. Mass spectrometer components

31. An ideal interface should:
► Quantitatively transfer all analyte
► Reduce pressure/flow from chromatograph to level
that MS can handle
► No interface meets all requirements.
The interface

32. Interfacing

33. ► The major goal of the interface is to remove most of the
carrier gas - the majority of the effluent.
► Classical or Molecular jet separator
► Permeation separator
► Open split
► Capillary direct

34. Molecular (Ryhage) jet Separator
The classical jet separator or molecular jet interface for
GC/MS was developed from the original Becker jet separator.

35. In these separators, the GC flow is introduced into an evacuated chamber
through a restricted capillary. At the capillary tip a supersonic expanding
jet of analyte and carrier molecules is formed and its core area sampled
into the mass spectrometer.
In an expanding jet, high molecular mass compounds are concentrated in
the core flow whereas the lighter and more diffusive carrier molecules are
dispersed away, in part through collisions.
Thus, sampling of the core flow produces an enrichment of the analyte.
The jet interface is very versatile, inert and efficient, despite
disadvantages of reduced efficiency with more volatile compounds and
potential plugging problems at the capillary restrictor.
Advantage: Relative simple and inexpensive approach.
Disadvantages :Rate of diffusion is MW dependent
selectivity based on MW

36. Permeation interface: Llwellyn Littlejohn separator
The permselective membrane interface, developed by Llewellyn and
Littlejohn is made of a silicone-rubber membrane that transmits
organic non-polar molecules and acts as a barrier for (non-organic)
carrier gases.
Despite being a very effective enrichment procedure, it also suffers
from discrimination effects with more polar analytes and produces
significant band broadening of their chromatographic peaks.

37. Major problems with this approach
1. Membrane selectivity based on polarity and MW
2. Slow to respond
3. Only a small fraction of analyte actually permeates
through membrane
Watson-Biemann effusion separator

38. The molecular effusion (or Watson Biemann) interface is based on
the molecular filtering of the gas effluent by means of a porous glass
frit. The column effluent passes through a fritted tube situated in a
vacuum chamber.
Small molecules traverse the microscopic pores in the tube walls and
are evacuated whereas high molecular mass molecules are
transferred to the ion source.
Among the principal drawbacks of this interface are the high dead
volume added and its high surface area. Also, as in the case of the jet
separator, this interface shows discrimination effects in the case of
smaller molecules.
The three methods presented above are based on the enrichment of the
analyte in the carrier gas by eliminating carrier molecules. In this way,
enough sample can be introduced into the ion source with total gas
flows compatible with the pumping capacity of the system.
Among them, the jet separator has been the
most extensively used and is perhaps the most successful interface.

39. The simple alternative to reduced total gas flow is flow
splitting.
In this case, no sample enrichment takes place and these procedures
are most useful where sensitivity is not a critical factor.
Flow splitting can be performed at the exit of the gas chromatograph,
allowing the diverted gas to be directed to a parallel detector, or at the
interface itself such as in the open split interface.
The latter is based on a capillary restrictor that limits the flow entering
the ion source to a manageable, constant value. The GC column exit is
situated close to the restrictor entrance in an open connector.
The restrictor samples the effluent from the GC column exit and the
excess column flow is removed from the connector by helium.
The open split interface is a versatile device that allows one to work
with a wide range of column flows without any interface modification.

40. Somewhat similar to a jet separator.
The MS pulls in about 1 ml/min through the flow restrictor.
If column flow is above that - excess is vented.
If flow is below that, He from external source is pulled in.
Best for sources that have flows close to 1 ml/min like capillary columns.
Advantages
1. Any gas producing source can be used.
2. Relative low cost and easy to use.
Disadvantages
1. Sample leaves column in split.
2. Split changes as flow changes.
Open split interface

41. Capillary direct interface
Advantages
1 Low cost, simple device
2 No dead volume
3 No selectivity
Disadvantages
1 Limits flow range that column can use
2 Limits the ID of column that can be used
3 Part of column is ‘lost’ - serves as a flow
restrictor.

42. HPTLC-MS interface
Interface
Mass
Analyzer
TLC

43. Advantages of TLC-MS system
1. Direct coupling of TLC plate with MS
without any pretreatment of the
separated chromatographic bands
2. A possibility of coupling a TLC plate
with a variety of MS types
3. A possibility of selecting individual
bands of interest for MS analysis
4. Identification of individual bands with
MS

44. TLC MS interface

45. The interface has the advantage that without
modification it can be integrated into any HPLC-MS
system featuring atmospheric pressure chemical
ionization (APCI), atmospheric pressure photo
ionisation (APPI), or electro spray ionization (ESI).
With two fittings the interface is connected to the
HPLC pump and the mass spectrometer. The
substance of interest is eluted directly from the
TLC/HPTLC plate and is transferred online into the
mass spectrometer.
Within a minute the mass spectrum is obtained.

46. Principle
► The versatile instrument is used to isolate unknown
compounds from a TLC/HPTLC plate and transfer them into
a mass spectrometer for identification or structure
elucidation. CAMAG TLC-MS Interface can be connected to
any brand of LC-coupled mass spectrometer.
► Plug & play installation by two HPLC fittings at a given
HPLC-MS system
► Semi-automatic instrument involving automatic piston
movement for pressure seal of the TLC/HPTLC zone on both
glass plates and aluminum foils
► Extraction directly from the plate using a suitable solvent
delivered by the HPLC pump
► Online transfer into the mass spectrometer
► Automatic cleaning of the piston between the extractions

47. The extraction principle
► Component mixtures, even with heavy matrix
load, can be separated cost efficiently on
TLC/HPTLC plates or aluminium foils.
► If the target zone is not visible, it can be
marked either under UV 254 nm or UV 366 nm,
by extrapolation of the adjacent zone made
visible by derivatization, or by using the hRF-
value obtained by TLC Scanner 3.
► By means of a laser crosshairs the zone to be
extracted is positioned exactly under the
extraction piston of the interface.
► The TLC-MS Interface is operated in semi-
automatic mode, which means that after
manual positioning of the zone the piston is
lowered at the push of a button.
► Moving a lever starts the solvent flow through
the layer and extracts the zone.

49. TLC- ESI-MS

51. TLC-Fast atom/ion bombardment-MS
TLC-APCI-MS

52. Example
► For identification of the zone at hRF 15 in a standard mixture of
caffeine, paracetamol and acetylsalicylic acid the mass spectrum of the
zone is recorded.
► At the same position a background spectrum of the plate is recorded
and subtracted from the substance spectrum.
► This leads to a mass spectrum free from system peaks showing mainly
substance signals - here the mass signal m/z 195 [M+H]+ for caffeine.
Left: Chromatogram with 4
mm bands, middle:
Same plate after extraction of
zone at hRF 15,
right: Extracted zone identified
as caffeine based on the mass
signal at m/z 195